METHODS AND COMPOSITIONS FOR TREATING CHRONIC INFLAMMATORY INJURY, METAPLASIA, DYSPLASIA AND CANCERS OF EPITHELIAL TISSUES
20230233691 · 2023-07-27
Assignee
Inventors
- Wa XIAN (Sugar Land, TX, US)
- Frank MCKEON (Sugar Land, TX, US)
- Shan WANG (Houston, TX, US)
- Audrey-Ann LIEW (Houston, TX, US)
- Matthew VINCENT (Amesbury, MA, US)
Cpc classification
A61K47/55
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A61K31/5025
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
A61K9/0024
HUMAN NECESSITIES
International classification
A61K47/55
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
A61K31/5025
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present disclosure provides methods and formulations for treating a patient suffering from one or more of chronic inflammatory injury, metaplasia, dysplasia or cancer of esophageal tissue and gastric tissue, which method comprises administering to the patient an agent that selectively kills or inhibits the proliferation or differentiation of pathogenic Barrett's Esophagus stem cells (BESCs) or Gastric Intestinal Metaplasia stem cells (GIMSCs) relative to normal regenerative esophageal stem cells or gastric stem cells in the tissue in which the BESCs or GIMSCs are found.
Claims
1. A method for treating a patient presenting with one or more of chronic inflammatory injury, metaplasia, dysplasia or cancer of an esophageal tissue, which method comprises administering to the patient an IAP Inhibitor agent that selectively kills or inhibits the proliferation or differentiation of pathogenic esophageal stem cells (PESC) in the esophageal tissue relative to normal regenerative stem cells of the epithelial tissue.
2. A method of reducing proliferation, survival, migration, or colony formation ability of a pathogenic esophageal stem cell (PESC) in a subject in need thereof comprising contacting the cell with a therapeutically effective amount of an IAP Inhibitor agent that selectively kills or inhibits the proliferation or differentiation of PESC relative to normal regenerative esophageal stem cells.
3. A pharmaceutical preparation for treating one or more of chronic inflammatory injury, metaplasia, dysplasia or cancer of an epithelial tissue, which preparation comprises an IAP Inhibitor agent that selectively kills or inhibits the proliferation or differentiation of pathogenic esophageal stem cells (PESC) in the esophageal tissue relative to normal regenerative esophageal stem cells.
4. (canceled)
5. A method for treating a patient presenting with one or more of esophagitis, Barrett's esophagus, esophageal dysplasia, esophageal cancer, gastric intestinal metaplasia or gastric cancer, which method comprises administering to the patient an IAP Inhibitor agent that selectively kills or inhibits the proliferation or differentiation of Barrett's Esophagus stem cells (BESC), gastric intestinal metaplasia (GIM) stem cells, esophageal cancer cells or gastric cancer cells relative to normal esophageal stem cells or stomach stem cells.
6. A method of reducing proliferation, survival, migration, or colony formation ability of a Barrett's Esophagus stem cell (BESC), gastric intestinal metaplasia (GIM) stem cells, esophageal cancer cells and gastric cancer cells in a subject in need thereof comprising contacting the cell with a therapeutically effective amount of an IAP Inhibitor agent that selectively kills or inhibits the proliferation or differentiation of BESC, GIM stem cells, esophageal cancer cells or gastric cancer cells relative to normal esophageal stem cells or stomach stem cells.
7. A pharmaceutical preparation for treating one or more of esophagitis, Barrett's esophagus, esophageal dysplasia esophageal cancer, gastric intestinal metaplasia, or gastric cancer, which preparation comprises an IAP Inhibitor agent that selectively kills or inhibits the proliferation or differentiation of Barrett's Esophagus stem cells (BESC), GIM stem cells, esophageal cancer cells or gastric cancer cells relative to normal esophageal stem cells or stomach stem cells.
8. A drug eluting device for treating one or more of esophagitis, Barrett's esophagus, esophageal dysplasia esophageal cancer, gastric intestinal metaplasia, or gastric cancer, which device comprises drug release means including an IAP Inhibitor agent that selectively kills or inhibits the proliferation or differentiation of Barrett's Esophagus stem cells (BESC), gastric intestinal metaplasia stem cells, esophageal cancer cells, or gastric cancer cells relative to normal esophageal stem cells or stomach stem cells, which device when deployed in a patient positions the drug release means proximal to the luminal surface of the esophagus or in the stomach region and releases the agent in an amount sufficient to achieve a therapeutically effective exposure of the luminal surface to the agent.
9. The method of claim 5 for the treatment of Barrett's Esophagus, Gastric Intestinal Metaplasia, esophageal adenocarcinoma, or gastric cancer.
10. (canceled)
11. The method of claim 5, wherein the IAP Inhibitor agent is administered during or after endoscopic ablation therapy, such as radiofrequency ablation, photodynamic therapy or cryoablation of esophageal tissue and gastric tissue.
12. The method of claim 5, wherein the IAP Inhibitor agent is administered by submucosal injection of esophageal tissue and gastric tissue.
13. (canceled)
14. The method of claim 5, wherein the IAP Inhibitor agent is formulated as part of a bioadhesive formulation.
15. The method of claim 5, wherein the IAP Inhibitor agent is formulated as part of a drug-eluting particle, drug eluting matrix or drug-eluting gel.
16. The method of claim 5, wherein the IAP Inhibitor agent is administered by topical application to the esophageal tissue and gastric tissue.
17-19. (canceled)
20. The method of claim 5, wherein the IAP Inhibitor agent is co-administered with an analgesic, an anti-infective or both.
21. (canceled)
22. The preparation of claim 7, wherein the IAP Inhibitor agent is formulated as a liquid for oral delivery to the epithelial tissue, such as the esophagus and stomach.
23. (canceled)
24. The device of claim 4, wherein the drug eluting device is a drug eluting stent or balloon catheter having a surface coating including the IAP Inhibitor agent.
25-26. (canceled)
27. The method of claim 5, wherein the IAP Inhibitor agent inhibits the proliferation or differentiation of BESCs, or kills BESCs, with an IC.sub.50 of 10.sup.−6 M or less, more preferably 10.sup.−7 M or less, 10.sup.−8 M or less or 10.sup.−9M or less.
28. (canceled)
29. The method of claim 5, wherein the IAP Inhibitor agent is an XIAP Inhibitor.
30. The method of claim 5, wherein the IAP Inhibitor agent is SM-164 or AZD5582.
31. The method of claim 5, further comprising combining the agent with a second drug agent that selectively promotes proliferation of normal regenerative esophageal stem cells in the target with an EC.sub.50 at least 5 times more potent than for BESCs in the target tissue, more preferably with an EC.sub.50 10 times, 50 times, 100 times or even 1000 times more potent than for BESCs.
32. (canceled)
33. The method of claim 5, wherein the second drug agent is a TAK1 inhibitor or a RET inhibitor.
34. The method of claim 5, wherein the second drug agent is pan-inhibitor of ABL kinase inhibitor selected from the group consisting of imatinib, nilotinib, dasatinib, bosutinib and ponatinib or pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof, and is preferably ponatinib or pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof.
35. The method of claim 5, wherein the second drug agent is FLT kinase inhibitor selected from the group consisting of quizartinib (AC220), crenolanib (CP-868596), midostaurin (PKC-412), lestaurtinib (CEP-701), 4SC-203, TTT-3002, sorafenib (Bay-43-0006), Ponatinib (AP-24534), sunitinib (SU-11248), and/or tandutinib (MLN-0518), or (a) pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof, and is preferably quizartinib or pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof.
36. The method of claim 5, wherein the TAP inhibitor is a bivalent SMAC mimetic and the second agent is a TAK1 inhibitor or a RET inhibitor.
37. The method of claim 5, further comprising combining the agent with a one or more antitussives, antihistamines, antipyretics, analgesics, anti-infective agents and/or chemotherapeutic agents.
38. The method of claim 31, wherein the agent and the second agent are administered to the patient as separate formulations.
39. The method of claim 21, wherein the agent and the second agent are co-formulated together.
40. The method of claim 37, wherein the agent and the one or more antitussives, antihistamines, antipyretics, analgesics, anti-infective agents and/or chemotherapeutic agents are co-formulated together.
41-44. (canceled)
45. A drug eluting device comprising drug release means including an IAP Inhibitor agent, which device when deployed in a patient positions the drug release means proximal to a target epithelial tissue to be treated and releases the IAP Inhibitor agent in an amount sufficient to achieve a therapeutically effective exposure of the target tissue to the IAP Inhibitor agent.
46. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0164] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0165]
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[0169]
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DETAILED DESCRIPTION
[0174] In a recent effort to deconvolute the cellular and genetic heterogeneity of lesional biopsies, the inventors applied technology that enables the cloning of normal gastrointestinal stem cells to endoscopic biopsies of Barrett's esophagus. This work demonstrated that Barrett's esophagus is dependent on a discrete population of highly immature stem cells with immense proliferative potential for its regenerative growth, and that these stem cells differentiate to an intestinal metaplasia indistinguishable from Barrett's esophagus.
I. Overview
[0175] Barrett's Esophagus holds a pivotal position at the interface of cancer biology and patient care. Barrett's was first discovered in 1950's and associated with risk for adenocarcinoma in the 1970's. Barrett's has become a paradigm for precancerous lesions giving rise to progressively more advanced lesions in a process requiring many years supporting an overall escalation model whereby non-cancerous lesions undergo long-term processes of stochastic changes some of which yield more sinister and determinant transitions to low- and high-grade dysplasia which then rapidly and almost inexorably evolve to malignant disease. The recognition of the importance of preemptive therapies that target these premalignant lesions is the foundation of cancer prevention. If true, the clinical solution to preventing the onset of esophageal adenocarcinoma would be simple and direct: ablate Barrett's before it can evolve to more aggressive lesions.
[0176] The advance of the development of targeted therapies for Barrett's requires conceptual advance of the origin of Barrett's and the recognition of the existence of Barrett's stem cells. If the premalignant stages of EAC represent the only tractable solution to this disease, it is essential to solve the mystery of the origin of BE and develop new therapeutic strategies specifically targeting its stem cells. However, the ontogeny of BE has been an intriguing puzzle with various hypotheses involving transcommitment of esophageal squamous stem cells, migration from lower gastrointestinal sites, the reparative emergence of submucosal glands, dissemination from bone marrow. The inventors recently showed that BE originated from the opportunistic growth of residual embryonic cells pre-existing at gastroesophageal junction (Wang et al., Cell. 2011 Jun. 24; 145(7):1023-1035). In addition, using the ground state stem cell technology that enabled us to clone stem cells of the normal human gastrointestinal tract, the inventors demonstrated the existence of the stem cells in BE (Yamamoto et al., Nat Commun. 2016 Jan. 19; 7:10380) and suggested they are the key elements to target in a therapeutic program designed to prevent the development and progression of this irreversible and dangerous metaplasia.
[0177] In order to uncover drugs specifically targeting BE stem cells that might synergize with physical ablation protocols to further reduce recurrent disease, provided herein is a multiplexed screening of established and experimental drugs or combinations thereof to identify compounds and combinations of compounds that selectively target the particular pathways that dominate the survival of these BE lesions. These BE stem cells were used in hybrid models with normal epithelial squamous stem cells to model the potential ability of such drug combinations to alter the competitive status of such lesions in the distal esophagus.
[0178] Also provided herein are screening methods that show the similar selective vulnerabilities of the stem cells of patient-matched BE, dysplasia and EAC, which suggest the broad usage of the pharmacological compositions that would augment physical ablation or mucosal dissection therapies. Indeed, as demonstrated by the data presented herein, the differential sensitivity of the pathogenic stem cells to single agents or combination therapies is carried across multiple tissues and across metaplasia, dysplasia or tumor samples from those tissues.
II. Definitions
[0179] Unless otherwise stated, the following terms used in the specification and claims are defined for the purposes of this Application and have the following meaning:
[0180] A “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as formic acid, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference. The compounds of the present disclosure can also exist as cocrystals.
[0181] The compounds of the present disclosure may have asymmetric centers. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active, racemic forms or other mixtures of isomers. It is well known in the art how to prepare optically active forms, such as by resolution of materials. All chiral, diastereomeric, racemic forms are within the scope of this disclosure, unless the specific stereochemistry or isomeric form is specifically indicated.
[0182] Certain compounds of can exist as tautomers and/or geometric isomers. All possible tautomers and cis and trans isomers, as individual forms and mixtures thereof are within the scope of this disclosure. Additionally, as used herein the term alkyl includes all the possible isomeric forms of said alkyl group albeit only a few examples are set forth.
[0183] A “pharmaceutically acceptable carrier or excipient” means a carrier or an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier or an excipient that is acceptable for veterinary use as well as human pharmaceutical use. “A pharmaceutically acceptable carrier/excipient” as used in the specification and claims includes both one and more than one such excipient.
[0184] “Substitution”. As described herein, compounds of the disclosure may contain optionally substituted and/or substituted moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
[0185] “Treating” or “treatment” of a disease includes: preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease; inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
III. Exemplary Embodiments
[0186] A. IAP Inhibitor
[0187] IAP (Inhibitor of apoptosis) proteins, a family of anti-apoptotic proteins, have an important role in evasion of apoptosis, as they can both block apoptosis-signaling pathways and promote survival. Eight members of this family have been described in humans (BIRC1/NAIP, BIRC2/cIAP1, BIRC3/cIAP2, BIRC4/XIAP, BIRC5/Survivin, BIRC6/Apollon, BIRC7/ML-IAP and BIRC8/ILP2). In certain embodiments, the agent is an IAP Inhibitor (i.e., an IAP Antagonist). Exemplary IAP Inhibitors include XIAP inhibitors, CIAP inhibitors, and agents acting as dual XIAP and CIAP inhibitors.
[0188] Exemplary IAP inhibitors and antagonists include Birinapant (a bivalent Smac mimetic, which is a potent antagonist for XIAP and cIAP1 with Kds of 45 nM and less than 1 nM, respectively), LCL161 Inhibitor (an IAP inhibitor which inhibits XIAP and cIAP1 with IC.sub.50s of 35 and 0.4 nM), AZD5582 (AZD5582 an IAP antagonist which binds to the BIR3 domains cIAP1, cIAP2, and XIAP), SM-164 (a cell-permeable Smac mimetic compound that binds to XIAP protein containing both the BIR2 and BIR3 domains with an IC.sub.50 value of 1.39 nM and functions as an extremely potent antagonist of XIAP), BV6 (an antagonist of cIAP1 and XIAP), Xevinapant (or AT-406, is a potent and orally bioavailable Smac mimetic and an antagonist of IAPs, and it binds to XIAP, cIAP1, and cIAP2 proteins), GDC-0152 (a potent IAPs inhibitor, and binds to the BIR3 domains of XIAP, cIAP1, cIAP2 and the BIR domain of ML-IAP), ASTX660 (an orally bioavailable dual antagonist of cIAPs and XIAPs), CUDC-427 (a potent second-generation pan-selective IAP antagonist), Embelin (or Embelic acid, a potent, nonpeptidic XIAP inhibitor). APG-1387 (a bivalent SMAC mimetic and an IAP antagonist, blocks the activity of IAPs family proteins (XIAP, cIAP-1, cIAP-2, and ML-IAP), MX69 (an inhibitor of MDM2/XIAP), AEG40826 (HGS1029) MV1, Polygalacin D, UC-112, AZD5582 dihydrochloride, HY-125378m Tolinapant (ASTX660) and SBP-0636457. In some embodiments, exemplary IAP inhibitors and antagonists include those described in one or more of WO2011098904; WO2009136290; WO2007106192; WO2008014238; WO2008128121 WO2012080271; U.S. Pat. No. 8,202,902; WO2013103703; US20140303090; WO2022130411; WO2017117684 and WO2015092420.
[0189] In certain embodiments, the IAP inhibitor is a selective XIAP inhibitor (having an IC.sub.50 for XIAP inhibition at least 10-fold less than the IC.sub.50 for CIAP inhibition, and more preferably at least 20. 50 or 100-fold less), such as SM-164.
[0190] B. Combination Therapies—ESO Regenerative Agent
[0191] In certain embodiments, the IAP Inhibitor agent can be administered conjointly with one or more agents that selectively promote proliferation or other regenerative and wound healing activities of normal regenerative esophageal stem cells. Conjoint administration of these “ESO Regenerative agents” may be accomplished by administration of a single co-formulation, by simultaneous administration or by administration at separate times.
[0192] In certain embodiments, the IAP Inhibitor agent can be administered conjointly with one or more agents that selectively promote proliferation or other regenerative and wound healing activities of normal esophageal stem cells. Conjoint administration of these “esophageal ESO Regenerative agents” may be accomplished by administration of a single co-formulation, by simultaneous administration or by administration at separate times.
[0193] TAK1 Inhibitor. In certain embodiments, the IAP Inhibitor agent is administered conjointly with a TAK1 inhibitor.
[0194] “Transforming growth factor activated kinase-1” and “TAK1” are used interchangeably. TAK1 is a protein kinase of the MLK family that mediates signal transduction induced by TGF beta and morphogenetic protein (BMP) and controls a variety of cell functions including transcription regulation and apoptosis. An illustrative non-limitative example of TAK1 is the human TAK1 protein Uniprot database accession number 043318. A “TAK1 inhibitor” as used herein is an agent that reduces or prevents TAK1 activity.
[0195] Exemplary embodiments of TAK1 inhibitors include 5Z-7-oxozeaenol, 2-[(aminocarbonyl)amino]-5-[4-(morpholin-4-ylmethyl)phenyl]thiophene-3-carboxamide, 2-[(aminocarbonyl)amino]-5-[4-(1-piperidin-1-ylethyl)phenyl]thiophene-3-carboxamide, 3-[(aminocarbonyl)amino]-5-[4-(morpholin-4-ylmethyl)phenyl]thiophene-2-carboxamide, and 3-[(aminocarbonyl)amino]-5-(4-{[(2-methoxy-2-methylpropyl)amino]methyl}phenyl)thiophene carboxamide.
[0196] In still other embodiments, the TAK1 inhibitor is dehydroabietic acid, NG25 (CAS No. 1315355-93-1), sarsasapogenin, takinib, 1-(3-(tert-Butyl)-1-(3-cyanophenyl)-1H-pyrazol-5-yl)-3-(3-methyl-4-(pyridin-4-yloxy)phenyl)urea (PF-05381941 or CAS: 1474022-02-0), 5Z-7-′, TAK1-IN1, minnelide, triptolide or a pharmaceutically acceptable salt or mixture thereof.
[0197] In one aspect, provided is a compound according to Formula:
##STR00017##
[0198] or a stereoisomer or salt thereof;
[0199] wherein [0200] X is NR.sub.1 or S; [0201] R.sub.1 is H, C1-4 alkyl, C1-4 carbonyl, or C1-4 carboxyl; [0202] R.sub.2 is H, C1-4 alkyl, C1-4 alkoxy, or halogen; [0203] R.sub.3 is OH, C1-4 alkoxy, or amino; and [0204] R.sub.4 is H, C1-4 alkyl, C1-4 alkoxy, or halogen;
[0205] wherein each C1-4 alkyl may be independently substituted by halo, hydroxy, or amino;
[0206] In certain embodiments, the TAK1 inhibitor is Takinib, and has the chemical structure
##STR00018##
[0207] In certain embodiments, the TAK1 inhibitor is NG25, and has the chemical structure
##STR00019##
[0208] For example, the TAK1 inhibitor is 5Z-7-Oxozeaenol, having the structure:
##STR00020##
[0209] In certain embodiments, the TAK1 inhibitor is an inhibitor of autophosphorylated and non-phosphorylated TAK1 that binds within the ATP-binding pocket and inhibits by slowing down the rate-limiting step of TAK1 activation.
[0210] In certain embodiments, the TAK1 inhibitor is an ATP-competitive irreversible inhibitor of TAK1.
[0211] In certain embodiments, the TAK1 inhibitor has Ki of 10 μM or less for TAK1 as well as IRAK4, IRAK1, GCK, CLK2, and MINK1.
[0212] In certain embodiments, the TAK1 inhibitor has Ki for IRAK4, IRAK1, GCK, CLK2, and MINK1 that is at least 5 times greater than the Ki for TAK1, and even more preferably at least 10, 25, 50 or even 100 times greater.
[0213] In certain preferred embodiments, the TAK1 inhibitor has a half maximal inhibitory concentration (IC.sub.50) value of 100 nM or less, and even more preferably 50 nM, 25 nM or even 10 nM or less.
[0214] In certain embodiments, the TAK1 inhibitor induces TNF-α-dependent induction of apoptosis,
[0215] Alternatively, the TAK1 inhibitor is for example an antisense TAK1 nucleic acid, a TAK1 specific short-interfering RNA, or a TAK1-specific ribozyme. By the term “siRNA” is meant a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into a cell are used, including those in which DNA is a template from which an siRNA is transcribed. The siRNA includes a sense TAK1 nucleic acid sequence, an anti-sense TAK1 nucleic acid sequence or both. Optionally, the siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin (shRNA).
[0216] c-RET inhibitor. In certain embodiments, the IAP Inhibitor agent is administered conjointly with a RET inhibitor, i.e., an inhibitor or the proto-oncogene tyrosine-protein kinase receptor Ret, also known as Cadherin family member 12 or Proto-oncogene c-Ret; UniprotKB—P07949). For instance, reviews are published disclosing such RET kinase inhibitors (Roskoski et Sadeghi-Nejad, Pharmacol Res. 2018 February; 128:1-17; Zschabitz et Grüllich; Recent Results Cancer Res. 2018; 211:187-198; Grüllich, Recent Results Cancer Res. 2018; 211:67-75; Pitoia et Jerkovich, Drug Des Devel Ther. 2016 Mar. 11; 10:1119-31), the disclosure of which being incorporated herein by reference. Patent applications also disclose RET kinase inhibitors, for instance and non-exhaustively WO18071454, WO18136663, WO18136661, WO18071447, WO18060714, WO18022761, WO18017983, WO17146116, WO17161269, WO17146116, WO17043550, WO17011776, WO17026718, WO14050781, WO07136103, WO06130673, the disclosure of which being incorporated herein by reference.
[0217] In certain embodiments, the RET inhibitor is selected from the group consisting of AD80, Regorafenib (BAY 73-4506), Cabozantinib malate (XL184), Fedratinib (TG101348), Danusertib (PHA-739358), TG101209, Agerafenib (RXDX-105), Regorafenib Hydrochloride, Selpercatinib (LOXO-292), Pralsetinib (BLU-667), GSK3179106, Regorafenib (BAY-734506) Monohydrate, vandetanib, RXDX-105, lenvatinib, sorafenib, sunitinib, dovitinib, alectinib, ponatinib, regorafenib, nintedanib, apatinib, motesanib, BLU-667, or LOXO-292.
[0218] In certain embodiments, the RET inhibitor may be WHI-P180, Apatinib, CS-2660 (JNJ-38158471), 2-D08,
[0219] In certain embodiments, the RET inhibitor is AD80 and has the chemical structure ′
##STR00021##
[0220] In certain embodiments, the RET inhibitor has a half maximal inhibitory concentration (IC.sub.50) value of 100 nM or less, and even more preferably 50 nM, 25 nM, 10 nM or even 5 nM or less.
[0221] Alternatively, the RET inhibitor is for example an antisense RET nucleic acid, a RET specific short-interfering RNA, or a RET-specific ribozyme. By the term “siRNA” is meant a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into a cell are used, including those in which DNA is a template from which an siRNA is transcribed. The siRNA includes a sense RET nucleic acid sequence, an anti-sense RET nucleic acid sequence or both. Optionally, the siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin (shRNA).
[0222] ABL kinase inhibitor. In certain embodiments, the ESO Regenerative agent is pan-inhibitor of ABL kinase inhibitor, preferably a BCR-ABL kinase inhibitor. Exemplary pan-inhibitor include imatinib, nilotinib, dasatinib, bosutinib and ponatinib, and is preferably ponatinib.
[0223] FLT3 Inhibitors. In certain embodiments, the ESO Regenerative agent is a FLT3 inhibitor. Exemplary FLT3 inhibitors to be used herein are quizartinib (AC220), crenolanib (CP-868596), midostaurin (PKC-412), lestaurtinib (CEP-701), 4SC-203, TTT-3002, sorafenib (Bay-43-0006), Ponatinib (AP-24534), sunitinib (SU-11248), and/or tandutinib (MLN-0518), or (a) pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof. Preferably, the FMS-like tyrosine kinase 3 (FLT3) inhibitor is quizartinib (AC220) or pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof.
[0224] These and further exemplary inhibitors to be used herein are described in more detail below. [0225] Brand Name: Quizartinib
Structure:
[0226] ##STR00022##
Affinities: FLT3 (1.6 nM), KIT (4.8 nM), PDGFRB (7.7 nM), RET (9.9 nM), PDGFRA (11 nM), CSF1R (12 nM) [0227] Brand Name: Crenolanib
Structure:
[0228] ##STR00023##
Affinities: FLT3, PDGFRb
[0229] Brand Name: Midostaurin
Structure:
[0230] ##STR00024##
Affinities: PKN1 (9.3 nM), TBK1 (9.3 nM), FLT3 (11 nM), JAK3 (12 nM), MLK1 (15 nM), and 30 targets in the range 15-110 nM [0231] Brand Name: Lestaurtinib
##STR00025##
Affinities: FLT3, TRKA, TRKB, TRKC
[0232] Brand Name: 4SC-203
Structure:
[0233] ##STR00026##
Affinities: FLT3, VEGFR
Structure:
[0234] ##STR00027##
Affinities: FLT3 (Wall, Blood (ASH Annual Meeting Abstracts). 2012; 120:866);
[0235] LRRK2 (Yao, Human molecular genetics. 2013; 22(2):328-44).
Clinical Phase: Preclinical
[0236] Developer: Tautatis (originator) [0237] Brand Name: Sorafenib
Code Name: Bay-43-0006
Structure:
[0238] ##STR00028##
IUPAC Name: 4-[4-[3-[4-Chloro-3-(trifluoromethyl)phenyl]ureido]phenoxy]-N-methylpyridine-2-carboxamide
Affinities: DDR1 (1.5 nM), HIPK4 (3 nM), ZAK (6 nM), DDR2 (7 nM), FLT3 (13 nM), and 15 targets in the range 13-130 nM (Zarrinkar, Gunawardane et al. 2009, loc. cit.) Clinical Phase: Launched (renal and hepatocellular carcinoma), Phase I/O (blood cancer) Developer: Bayer [0239] Brand Name: Ponatinib
Code Name: AP-24534 Structure:
[0240] ##STR00029##
IUPAC Name: 3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-[4-(4-methylpiperazin-1-ylmethyl)-3-(trifluoromethyl)phenyl]benzamide
Affinities: BCR-ABL, FLT3, KIT, FGFR1, PDGFRa (Gozgit, Mol Cancer Ther. 2011; 10(6):1028-35).
Clinical Phase: Phase II (AML)
[0241] Developer: Ariad Pharmaceuticals (originator) [0242] Brand Name: Sunitinib
Code Name: SU-11248
Structure:
[0243] ##STR00030##
IUPAC Name: (Z)—N-[2-(Diethylamino)ethyl]-5-(5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide 2(S)˜hydroxybutanedioic acid (1:1) N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide L-malate
Affinities: PDGFRB (0.075 nM), KIT (0.37 nM), FLT3 (0.47 nM), PDGFRA (0.79 nM), DRAK1 (1.0 nM), VEGFR2 (1.5 nM), FLT1 (1.8 nM), CSF1R (2.0 nM) (Zarrinkar, Gunawardane et al. 2009, loc. cit.)
Clinical Phase: Launched (renal cell carcinoma, gastrointestinal stromal cancer, neuroendocrine pancreas), phase I (AML)
Developer: Pfizer (Originator)
[0244] Brand Name: Tandutinib
Code Name: MLN-0518
Structure:
[0245] ##STR00031##
IUP AC Name: N-(4-Isopropoxyphenyl)-4-[6-methoxy-7-[3-(1-piperidinyl)propoxy]quinazolin-4-yl]piperazine-1-carboxamide
Affinities: PDGFRA (2.4 nM), KIT (2.7 nM), FLT3 (3 nM), PDGFRB (4.5 nM), CSF1R (4.9 nM) (Zarrinkar, Gunawardane et al. 2009, loc. cit.)
Clinical Phase: discontinued
Developer: Kyowa Hakko Kirin (Originator), Millennium Pharmaceuticals (Originator),
[0246] Code Name: FF-10101
Structure:
[0247] ##STR00032##
[0248] National Cancer Institute, Takeda (Originator) FLT3 inhibitors to be used in accordance with the present disclosure are not limited to the herein described or further known exemplary inhibitors. Accordingly, also further inhibitors or even yet unknown inhibitors may be used in accordance with the present disclosure. Such inhibitors may be identified by the methods described and provided herein and methods known in the art, like high-throughput screening using biochemical assays for inhibition of FLT3.
[0249] Assays for screening potential FLT3 inhibitors and, in particular, for identifying FLT3 inhibitors as defined herein, comprise, for example, in vitro competition binding assays to quantitatively measure interactions between test compounds and recombinantly expressed kinases.sup.1 (Fabian et al; Nat Biotechnol. 2005 23(3):329-36). Hereby, competition with immobilized capture compounds and free test compounds is performed. Test compounds that bind the kinase active site will reduce the amount of kinase captured on solid support, whereas test molecules that do not bind the kinase have no effect on the amount of kinase captured on the solid support. Furthermore, inhibitor selectivity can also be assessed in parallel enzymatic assays for a set of recombinant protein kinases. (Davies et al., Biochem. J. 2000 35(1): 95-105; Bain et al. Biochem. J. 2003 37(1): 199-204). These assays are based on the measurement of the inhibitory effect of a kinase inhibitor and determine the concentration of compound required for 50% inhibition of the protein kinases of interest. Proteomics methods are also an efficient tool to identify cellular targets of kinase inhibitors. Kinases are enriched from cellular lysates by immobilized capture compounds, so the native target spectrum of a kinase inhibitor can be determined..sup.4 (Godl et al., Proc Natl Acad Sci USA. 2003 100(26): 5434-9).
[0250] Assays for screening of potential inhibitors and, in particular, for identifying inhibitors as defined herein, are, for example, described in the following papers: [0251] FABIAN ET AL., NAT BIOTECHNOL. 2005 23(3):329-36 [0252] DAVIES ET AL., BIOCHEM. J. 2000 351: 95-105. [0253] BAIN ET AL., BIOCHEM. J. 2003 371: 199-204. [0254] GODL ET AL., PROC NATL ACAD SCI USA. 2003 100(26): 15434-9.
The above papers are incorporated herein in their entirety by reference.
IV. Combination Therapies—Other Agents
[0255] In certain embodiments, the IAP Inhibitor agent can be administered conjointly with one or more agents that have other beneficial local activities in esophagus. Illustrative categories and specific examples of active drugs include: (a) antitussives, such as dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, and chlophedianol hydrochloride; (b) antihistamines, such as chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate; (c) antipyretics and analgesics such as acetaminophen, aspirin and ibuprofen; (d) antacids such as aluminum hydroxide and magnesium hydroxide, (e) anti-infective agents such as antifungals, antivirals, antiseptics and antibiotics, (f) chemotherapeutic agents.
V. Exemplary Formulations
[0256] In certain embodiments, the IAP Inhibitor agents is formulated for topical administration as part of a bioadhesive formulation. Bioadhesive polymers have extensively been employed in transmucosal drug delivery systems and can be readily adapted for use in delivery of the subject IAP Inhibitor agents to the esophagus, particularly the areas of lesions and tumor growth. In general terms, adhesion of polymers to tissues may be achieved by (i) physical or mechanical bonds, (ii) primary or covalent chemical bonds, and/or (iii) secondary chemical bonds (i.e., ionic). Physical or mechanical bonds can result from deposition and inclusion of the adhesive material in the crevices of the mucus or the folds of the mucosa. Secondary chemical bonds, contributing to bioadhesive properties, consist of dispersive interactions (i.e., Van der Waals interactions) and stronger specific interactions, which include hydrogen bonds. The hydrophilic functional groups responsible for forming hydrogen bonds are the hydroxyl (—OH) and the carboxylic groups (—COOH). When these materials are incorporated into pharmaceutical formulations, drug absorption by mucosal cells may be enhanced and/or the drug may be released at the site for an extended period of time. Merely to illustrate, the bioadhesive can be a hydrophilic polymer, a hydrogel, a co-polymers/interpolymer complex or a thiolated polymer. [0257] Hydrophilic polymers: these are water-soluble polymers that swell when they come in contact with water and eventually undergo complete dissolution. Systems coated with these polymers show high bioadhesiveness to the mucosa in dry state but the bioadhesive nature deteriorates as they start dissolving. As a result, their bioadhesiveness is short-lived. An example is poly (acrylic acid). [0258] Hydrogels: these are three-dimensional polymer networks of hydrophilic polymers which are cross-linked either by chemical or physical bonds. These polymers swell when they come in contact with water. The extent of swelling depends upon the degree of crosslinking. Examples are polycarbophil, carbopol and polyox. [0259] Co-polymers/interpolymer complex: a block copolymer is formed when the reaction is carried out in a stepwise manner, leading to a structure with long sequences or blocks of one monomer alternating with long sequences of the other. There are also graft copolymers, in which entire chains of one kind (e.g., polystyrene) are made to grow out of the sides of chains of another kind (e.g., polybutadiene), resulting in a product that is less brittle and more impact-resistant. Hydrogen bonding is a major driving force for interpolymer interactions. [0260] Thiolated polymers (thiomers): these are hydrophilic macromolecules exhibiting free thiol groups on the polymeric backbone. Based on thiol/disulfide exchange reactions and/or a simple oxidation process disulfide bonds are formed between such polymers and cysteine-rich subdomains of mucus glycoproteins building up the mucus gel layer. So far, the cationic thiomers, chitosan-cysteine, chitosan-thiobutylamidine as well as chitosan-thioglycolic acid, and the anionic thiomers, poly (acylic acid)-cysteine, poly (acrylic acid)-cysteamine, carboxymethylcellulose-cysteine and alginate-cysteine, have been generated. Due to the immobilisation of thiol groups on mucoadhesive basis polymers, their mucoadhesive properties are 2- up to 140-fold improved.
[0261] In certain embodiments, the bioadhesive polymer can be selected from poly(acrylic acid), tragacanth, poly(methylvinylether comaleic anhydride), poly(ethylene oxide), methyl-cellulose, sodium alginate, hydroxypropylmethylcellulose, karaya gum, methylethyl cellulose (and cellulose derivatives such as Metolose), soluble starch, gelatin, pectin, poly(vinyl pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(hydroxyethyl-methacrylate), hydroxypropylcellulose, sodium carboxymethylcellulose or chitosan.
[0262] Other suitable bioadhesive polymers are described in U.S. Pat. No. 6,235,313 to Mathiowitz et al., the teachings of which are incorporated herein by reference, and include polyhydroxy acids, such as poly(lactic acid), polystyrene, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan; polyacrylates, such as poly(methyl methacrylates), poly(ethyl methacrylates), poly butylmethacrylate), poly-(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyacrylamides; poly(fumaric-co-sebacic)acid, poly(bis carboxy phenoxy propane-co-sebacic anhydride), polyorthoesters, and copolymers, blends and mixtures thereof.
[0263] In certain embodiments, the bioadhesive is an alginate. Alginic acid and its salts associates with sodium and potassium bicarbonate have shown that, after entering a more acidic environment they form a viscous suspension (or a gel) exerting protecting activity over gastric mucosa. These properties are readily adaptable for topical delivery to the esophagus, particularly the lower esophagus. The scientific and patent literature on its activity is wide. Thus, for example, for delivery to the esophagus: Mandel K. G.; Daggy B. P.; Brodie D. A; Jacoby, H. L., 2000. Review article: Alginate-raft formulations in the treatment of heartburn and acid reflux. Aliment. Pharmacol. Ther. 14 669-690, which is incorporated by reference herein in its entirety; and Bioadhesive esophageal bandages: protection against acid and pepsin injury. Man Tang, Peter Dettmar, Hannah Batchelor—International Journal of Pharmaceutics 292 (2005)-169-177, which is incorporated by reference herein in its entirety.
[0264] In certain embodiments, the bioadhesive is a bioadhesive hydrogel. Bioadhesive hydrogels are well known in art and suitable hydrogels that be used for delivery of the IAP Inhibitor agents of the present disclosure are described in a wide range of scientific and patent literature on its activity is wide. An exemplary hydrogel formulation is described in Collaud et al. “Clinical evaluation of bioadhesive hydrogels for topical delivery of hexylaminolevulinate to Barrett's esophagus” J Control Release. 2007 Nov. 20; 123(3):203-10.
[0265] Bioadhesive Microparticle formulations. In certain embodiments, the IAP Inhibitor agent (optionally with other active agents) are formulated into adhesive polymeric microspheres have been selected on the basis of the physical and chemical bonds formed as a function of chemical composition and physical characteristics, such as surface area, as described in detail below. These microspheres are characterized by adhesive forces to mucosa of greater than 11 mN/cm.sup.2 on esophageal tissue. The size of these microspheres can range from between a nanoparticle to a millimeter in diameter. The adhesive force is a function of polymer composition, biological substrate, particle morphology, particle geometry (e.g., diameter) and surface modification.
[0266] Suitable polymers that can be used to form bioadhesive microspheres include soluble and insoluble, biodegradable and nonbiodegradable polymers. These can be hydrogels or thermoplastics, homopolymers, copolymers or blends, natural or synthetic. The preferred polymers are synthetic polymers, with controlled synthesis and degradation characteristics. Most preferred polymers are copolymers of fumaric acid and sebacic acid, which have unusually good bioadhesive properties when administered to the gastrointestinal.
[0267] In the past, two classes of polymers have appeared to show useful bioadhesive properties: hydrophilic polymers and hydrogels. In the large class of hydrophilic polymers, those containing carboxylic groups (e.g., poly[acrylic acid]) exhibit the best bioadhesive properties. One could infer that polymers with the highest concentrations of carboxylic groups should be the materials of choice for bioadhesion on soft tissues. In other studies, the most promising polymers were sodium alginate, carboxymethylcellulose, hydroxymethylcellulose and methylcellulose. Some of these materials are water-soluble, while others are hydrogels.
[0268] Rapidly bioerodible polymers such as poly[lactide-co-glycolide], polyanhydrides, and polyorthoesters, whose carboxylic groups are exposed on the external surface as their smooth surface erodes, are excellent candidates for bioadhesive drug delivery systems. In addition, polymers containing labile bonds, such as polyanhydrides and polyesters, are well known for their hydrolytic reactivity. Their hydrolytic degradation rates can generally be altered by simple changes in the polymer backbone.
[0269] Representative natural polymers include proteins, such as zein, modified zein, casein, gelatin, gluten, serum albumin, or collagen, and polysaccharides, such as cellulose, dextrans, polyhyaluronic acid, polymers of acrylic and methacrylic esters and alginic acid. These are not preferred due to higher levels of variability in the characteristics of the final products, as well as in degradation following administration. Synthetically modified natural polymers include alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and nitrocelluloses.
[0270] Representative synthetic polymers include polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof. Other polymers of interest include, but are not limited to, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone, and polyvinylphenol. Representative bioerodible polymers include polylactides, polyglycolides and copolymers thereof, poly(ethylene terephthalate), poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), poly[lactide-co-glycolide], polyanhydrides, polyorthoesters, blends and copolymers thereof.
[0271] These polymers can be obtained from sources such as Sigma Chemical Co., St. Louis, Mo., Polysciences, Warrenton, Pa., Aldrich, Milwaukee, Wis., Fluka, Ronkonkoma, N.Y., and BioRad, Richmond, Calif. or else synthesized from monomers obtained from these suppliers using standard techniques.
[0272] In some instances, the polymeric material could be modified to improve bioadhesion either before or after the fabrication of microspheres. For example, the polymers can be modified by increasing the number of carboxylic groups accessible during biodegradation, or on the polymer surface. The polymers can also be modified by binding amino groups to the polymer. The polymers can also be modified using any of a number of different coupling chemistries that covalently attach ligand molecules with bioadhesive properties to the surface-exposed molecules of the polymeric microspheres.
[0273] One useful protocol involves the “activation” of hydroxyl groups on polymer chains with the agent, carbonyldiimidazole (CDI) in aprotic solvents such as DMSO, acetone, or THF. CDI forms an imidazolyl carbamate complex with the hydroxyl group which may be displaced by binding the free amino group of a ligand such as a protein. The reaction is an N-nucleophilic substitution and results in a stable N-alkylcarbamate linkage of the ligand to the polymer. The “coupling” of the ligand to the “activated” polymer matrix is maximal in the pH range of 9-10 and normally requires at least 24 hrs. The resulting ligand-polymer complex is stable and resists hydrolysis for extended periods of time.
[0274] Another coupling method involves the use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or “water-soluble CDI” in conjunction with N-hydroxylsulfosuccinimide (sulfo NHS) to couple the exposed carboxylic groups of polymers to the free amino groups of ligands in a totally aqueous environment at the physiological pH of 7.0. Briefly, EDAC and sulfo-NHS form an activated ester with the carboxylic acid groups of the polymer which react with the amine end of a ligand to form a peptide bond. The resulting peptide bond is resistant to hydrolysis. The use of sulfo-NHS in the reaction increases the efficiency of the EDAC coupling by a factor of ten-fold and provides for exceptionally gentle conditions that ensure the viability of the ligand-polymer complex.
[0275] By using either of these protocols it is possible to “activate” almost all polymers containing either hydroxyl or carboxyl groups in a suitable solvent system that will not dissolve the polymer matrix.
[0276] A useful coupling procedure for attaching ligands with free hydroxyl and carboxyl groups to polymers involves the use of the cross-linking agent, divinylsulfone. This method would be useful for attaching sugars or other hydroxylic compounds with bioadhesive properties to hydroxylic matrices. Briefly, the activation involves the reaction of divinylsulfone to the hydroxyl groups of the polymer, forming the vinylsulfonyl ethyl ether of the polymer. The vinyl groups will couple to alcohols, phenols and even amines. Activation and coupling take place at pH 11. The linkage is stable in the pH range from 1-8 and is suitable for transit through the intestine.
[0277] Any suitable coupling method known to those skilled in the art for the coupling of ligands and polymers with double bonds, including the use of UV crosslinking, may be used for attachment of bioadhesive ligands to the polymeric microspheres described herein. Any polymer that can be modified through the attachment of lectins can be used as a bioadhesive polymer for purposes of drug delivery or imaging.
[0278] Lectins that can be covalently attached to microspheres to render them target specific to the mucin and mucosal cell layer could be used as bioadhesives. Useful lectin ligands include lectins isolated from Abrus precatroius, Agaricus bisporus, Anguilla anguilla, Arachis hypogaea, Pandeiraea simplicifolia, Bauhinia purpurea, Caragan arobrescens, Cicer arietinum, Codiurn fragile, Datura stramonium, Dolichos biflorus, Erythrina corallodendron, Erythrina cristagalli, Euonymus europaeus, Glycine max, Helix aspersa, Helix pomatia, Lathyrus odoratus, Lens culinaris, Limulus polyphemus, Lysopersicon esculentum, Maclura pomifera, Momordica charantia, Mycoplasma gallisepticum, Naja mocambique, as well as the lectins Concanavalin A, Succinyl-Concanavalin A, Triticum vulgaris, Ulex europaeus I, II and III, Sambucus nigra, Maackia amurensis, Limax fluvus, Homarus americanus, Cancer antennarius, and Lotus tetragonolobus.
[0279] The attachment of any positively charged ligand, such as polyethyleneimine or polylysine, to any microsphere may improve bioadhesion due to the electrostatic attraction of the cationic groups coating the beads to the net negative charge of the mucus. The mucopolysaccharides and mucoproteins of the mucin layer, especially the sialic acid residues, are responsible for the negative charge coating. Any ligand with a high binding affinity for mucin could also be covalently linked to most microspheres with the appropriate chemistry, such as CDI, and be expected to influence the binding of microspheres to the gut. For example, polyclonal antibodies raised against components of mucin or else intact mucin, when covalently coupled to microspheres, would provide for increased bioadhesion. Similarly, antibodies directed against specific cell surface receptors exposed on the lumenal surface of the intestinal tract would increase the residence time of beads, when coupled to microspheres using the appropriate chemistry. The ligand affinity need not be based only on electrostatic charge, but other useful physical parameters such as solubility in mucin or else specific affinity to carbohydrate groups.
[0280] The covalent attachment of any of the natural components of mucin in either pure or partially purified form to the microspheres would decrease the surface tension of the bead-gut interface and increase the solubility of the bead in the mucin layer. The list of useful ligands would include but not be limited to the following: sialic acid, neuraminic acid, n-acetyl-neuraminic acid, n-glycolylneuraminic acid, 4-acetyl-n-acetylneuraminic acid, diacetyl-n-acetylneuraminic acid, glucuronic acid, iduronic acid, galactose, glucose, mannose, fucose, any of the partially purified fractions prepared by chemical treatment of naturally occurring mucin, e.g., mucoproteins, mucopolysaccharides and mucopolysaccharide-protein complexes, and antibodies immunoreactive against proteins or sugar structure on the mucosal surface.
[0281] The attachment of polyamino acids containing extra pendant carboxylic acid side groups, e.g., polyaspartic acid and polyglutamic acid, should also provide a useful means of increasing bioadhesiveness. Using polyamino acids in the 15,000 to 50,000 kDa molecular weight range would yield chains of 120 to 425 amino acid residues attached to the surface of the microspheres. The polyamino chains would increase bioadhesion by means of chain entanglement in mucin strands as well as by increased carboxylic charge.
[0282] As used herein, the term “microspheres” includes microparticles and microcapsules (having a core of a different material than the outer wall), having a diameter in the nanometer range up to 5 mm. The microsphere may consist entirely of bioadhesive polymer or have only an outer coating of bioadhesive polymer.
[0283] As characterized in the following examples, microspheres can be fabricated from different polymers using different methods. Polylactic acid blank microspheres were fabricated using three methods: solvent evaporation, as described by E. Mathiowitz, et al., J. Scanning Microscopy, 4, 329 (1990); L. R. Beck, et al., Fertil. Steril., 31, 545 (1979); and S. Benita, et al., J. Pharm. Sci., 73, 1721 (1984); hot-melt microencapsulation, as described by E. Mathiowitz, et al., Reactive Polymers, 6, 275 (1987); and spray drying. Polyanhydrides made of bis-carboxyphenoxypropane and sebacic acid with molar ratio of 20:80 P(CPP-SA) (20:80) (Mw 20,000) were prepared by hot-melt microencapsulation. Poly(fumaric-co-sebacic) (20:80) (Mw 15,000) blank microspheres were prepared by hot-melt microencapsulation. Polystyrene microspheres were prepared by solvent evaporation.
[0284] In certain embodiments, the composition includes a bioadhesive matrix in which particles (such as nanoparticles) containing the IAP Inhibitor agents are dispersed. In these embodiments, the bioadhesive matrix promotes contact between the mucosa of the esophagus and the nanoparticles.
[0285] In certain embodiments, the drug-containing particle is a matrix, such as a bioerodible, bioadhesive matrix. Suitable bioerodible, bioadhesive polymers include bioerodible hydrogels, such as those described by Sawhney, et al., in Macromolecules, 1993, 26:581-587, the teachings of which are incorporated herein by reference. Representative bioerodible, bioadhesive polymers include, but are not limited to, synthetic polymers such as poly hydroxy acids, such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide), poly(lactide-co-caprolactone), poly(ethylene-co-maleic anhydride), poly(ethylene maleic anhydride-co-L-dopamine), poly(ethylene maleic anhydride-co-phenylalanine), poly(ethylene maleic anhydride-co-tyrosine), poly(butadiene-co-maleic anhydride), poly(butadiene maleic anhydride-co-L-dopamine) (pBMAD), poly(butadiene maleic anhydride-co-phenylalanine), poly(butadiene maleic anhydride-co-tyrosine), poly(fumaric-co-sebacic)anhydride (P(FA:SA)), poly(bis carboxy phenoxy propane-co-sebacic anhydride) (20:80) (poly(CCP:SA)), as well as blends comprising these polymers; and copolymers comprising the monomers of these polymers, and natural polymers such as alginate and other polysaccharides, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers, blends and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
[0286] Particles having an average particle size of between 10 nm and 10 microns are useful in the compositions described herein. In certain embodiments, the particles are nanoparticles, having a size range from about 10 nm to 1 micron, preferably from about 10 nm to about 0.1 microns. In particularly preferred embodiments, the particles have a size range from about 500 to about 600 nm. The particles can have any shape but are generally spherical in shape.
[0287] The compositions described herein contain a monodisperse plurality of nanoparticles. Preferably, the method used to form the nanoparticles produces a monodisperse distribution of nanoparticles; however, methods producing polydisperse nanoparticle distributions can be used. If the method does not produce particles having a monodisperse size distribution, the particles are separated following particle formation to produce a plurality of particles having the desired size range and distribution.
[0288] Nanoparticles useful in the compositions described herein can be prepared using any suitable method known in the art. Common microencapsulation techniques include, but are not limited to, spray drying, interfacial polymerization, hot melt encapsulation, phase separation encapsulation (spontaneous emulsion microencapsulation, solvent evaporation microencapsulation, and solvent removal microencapsulation), coacervation, low temperature microsphere formation, and phase inversion nanoencapsulation (PIN). A brief summary of these methods is presented below.
[0289] Spray Drying. Methods for forming microspheres/nanospheres using spray drying techniques are described in U.S. Pat. No. 6,620,617, to Mathiowitz et al. In this method, the polymer is dissolved in an organic solvent such as methylene chloride or in water. A known amount of one or more active agents to be incorporated in the particles is suspended (in the case of an insoluble active agent) or co-dissolved (in the case of a soluble active agent) in the polymer solution. The solution or dispersion is pumped through a micronizing nozzle driven by a flow of compressed gas, and the resulting aerosol is suspended in a heated cyclone of air, allowing the solvent to evaporate from the microdroplets, forming particles. Microspheres/nanospheres ranging between 0.1-10 microns can be obtained using this method.
[0290] Interfacial Polymerization. Interfacial polymerization can also be used to encapsulate one or more active agents. Using this method, a monomer and the active agent(s) are dissolved in a solvent. A second monomer is dissolved in a second solvent (typically aqueous) which is immiscible with the first. An emulsion is formed by suspending the first solution through stirring in the second solution. Once the emulsion is stabilized, an initiator is added to the aqueous phase causing interfacial polymerization at the interface of each droplet of emulsion.
[0291] Hot Melt Microencapsulation. Microspheres can be formed from polymers such as polyesters and polyanhydrides using hot melt microencapsulation methods as described in Mathiowitz et al., Reactive Polymers, 6:275 (1987). In this method, the use of polymers with molecular weights between 3-75,000 daltons is preferred. In this method, the polymer first is melted and then mixed with the solid particles of one or more active agents to be incorporated that have been sieved to less than 50 microns. The mixture is suspended in a non-miscible solvent (like silicon oil), and, with continuous stirring, heated to 5° C. above the melting point of the polymer. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decanting with petroleum ether to give a free-flowing powder.
[0292] Phase Separation Microencapsulation. In phase separation microencapsulation techniques, a polymer solution is stirred, optionally in the presence of one or more active agents to be encapsulated. While continuing to uniformly suspend the material through stirring, a nonsolvent for the polymer is slowly added to the solution to decrease the polymer's solubility. Depending on the solubility of the polymer in the solvent and nonsolvent, the polymer either precipitates or phase separates into a polymer rich and a polymer poor phase. Under proper conditions, the polymer in the polymer rich phase will migrate to the interface with the continuous phase, encapsulating the active agent(s) in a droplet with an outer polymer shell.
[0293] Spontaneous Emulsion Microencapsulation. Spontaneous emulsification involves solidifying emulsified liquid polymer droplets formed above by changing temperature, evaporating solvent, or adding chemical cross-linking agents. The physical and chemical properties of the encapsulant, as well as the properties of the one or more active agents optionally incorporated into the nascent particles, dictates suitable methods of encapsulation. Factors such as hydrophobicity, molecular weight, chemical stability, and thermal stability affect encapsulation.
[0294] Solvent Evaporation Microencapsulation. Methods for forming microspheres using solvent evaporation techniques are described in E. Mathiowitz et al., Scanning Microscopy, 4:329 (1990); L. R. Beck et al., Fertil. Steril., 31:545 (1979); L. R. Beck et al., Am J Obstet Gynecol 135(3) (1979); S. Benita et al., Pharm. Sci., 73:1721 (1984); and U.S. Pat. No. 3,960,757 to Morishita et al. The polymer is dissolved in a volatile organic solvent, such as methylene chloride. One or more active agents to be incorporated are optionally added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid microspheres/nanospheres. This method is useful for relatively stable polymers like polyesters and polystyrene. However, labile polymers, such as polyanhydrides, may degrade during the fabrication process due to the presence of water. For these polymers, some of the following methods performed in completely anhydrous organic solvents are more useful.
[0295] Solvent Removal Microencapsulation. The solvent removal microencapsulation technique is primarily designed for polyanhydrides and is described, for example, in WO 93/21906 to Brown University Research Foundation. In this method, the substance to be incorporated is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent, such as methylene chloride. This mixture is suspended by stirring in an organic oil, such as silicon oil, to form an emulsion. Microspheres that range between 1-300 microns can be obtained by this procedure. Substances which can be incorporated in the microspheres include pharmaceuticals, pesticides, nutrients, imaging agents, and metal compounds.
[0296] Coacervation. Encapsulation procedures for various substances using coacervation techniques are known in the art, for example, in GB-B-929 406; GB-B-929 40 1; and U.S. Pat. Nos. 3,266,987, 4,794,000, and 4,460,563. Coacervation involves the separation of a macromolecular solution into two immiscible liquid phases. One phase is a dense coacervate phase, which contains a high concentration of the polymer encapsulant (and optionally one or more active agents), while the second phase contains a low concentration of the polymer. Within the dense coacervate phase, the polymer encapsulant forms nanoscale or microscale droplets. Coacervation may be induced by a temperature change, addition of a non-solvent or addition of a micro-salt (simple coacervation), or by the addition of another polymer thereby forming an interpolymer complex (complex coacervation).
[0297] Low Temperature Casting of Microspheres. Methods for very low temperature casting of controlled release microspheres are described in U.S. Pat. No. 5,019,400 to Gombotz et al. In this method, a polymer is dissolved in a solvent optionally with one or more dissolved or dispersed active agents. The mixture is then atomized into a vessel containing a liquid non-solvent at a temperature below the freezing point of the polymer-substance solution which freezes the polymer droplets. As the droplets and non-solvent for the polymer are warmed, the solvent in the droplets thaws and is extracted into the non-solvent, resulting in the hardening of the microspheres.
[0298] Phase Inversion Nanoencapsulation (PIN). Nanoparticles can also be formed using the phase inversion nanoencapsulation (PIN) method, wherein a polymer is dissolved in a “good” solvent, fine particles of a substance to be incorporated, such as a drug, are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non-solvent for the polymer, to spontaneously produce, under favorable conditions, polymeric microspheres, wherein the polymer is either coated with the particles or the particles are dispersed in the polymer. See, e.g., U.S. Pat. No. 6,143,211 to Mathiowitz, et al. The method can be used to produce monodisperse populations of nanoparticles and microparticles in a wide range of sizes, including, for example, about 100 nanometers to about 10 microns. Advantageously, an emulsion need not be formed prior to precipitation. The process can be used to form microspheres from thermoplastic polymers.
[0299] Sequential Phase Inversion Nanoencapsulation (sPIN). Multi-walled nanoparticles can also be formed by a process referred to herein as “sequential phase inversion nanoencapsulation” (sPIN). This process is described in detail below in Section IV. sPIN is particularly suited for forming monodisperse populations of nanoparticles, avoiding the need for an additional separations step to achieve a monodisperse population of nanoparticles.
[0300] Dissolving Tablets or Lozenges. In certain embodiments, the IAP Inhibitor agents is provided in a dissolving tablet. For example, the tablet can contain a therapeutically effective amount of the IAP Inhibitor agent in combination with polyvinylpyrrolidone (PVP: povidone), wherein the tablet is formulated to rapidly dissolve in a specific volume of liquid so as to generate a topical esophageal therapy suitable for delivering the anti-PESC to the luminal surface of the esophagus. For instance, the volume of liquid in which the tablet dissolves can be from 5 to 50 mL, 5 to 25 mL or even 5 to 15 mL. Preferably the liquid is water. The dissolving tablet can also further include an excipient that renders the dissolving tablet palatable, especially at least one excipient that increases viscosity of the topical esophageal therapy. An exemplary viscosity-enhancing excipient is mannitol.
[0301] In certain embodiments, the IAP Inhibitor agent is provided in a topical, non-systemic, oral, slow releasing, solid, soft lozenge pharmaceutical composition comprising: (a) about 1% to about 5% by mass of one or more release modifiers comprising polyethylene oxide polymers comprising a molecular weight of about 900,000 to about 8,000,000; (b) about 10% to about 60% by mass of one or more film-forming polymers comprising gelatins; (c) about 5% to about 20% by mass of one or more plasticizers comprising glycerol, sorbitol, or combinations thereof; and (d) less than 1% by mass of one or more IAP Inhibitor agents. Exemplary plasticizers include glycerol, sorbitol, mannitol, maltitol, xylitol, or combinations thereof. The lozenge may also include one or more sweeteners, such as maltitol, xylitol, mannitol, sucralose, aspartame, stevia, or a combination thereof. The lozenge may also include one or more pH modifiers comprising one or more organic acids.
VI. Examples
[0302] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Overview
[0303] The challenges presented by advanced metastatic cancer have driven efforts to identify and preemptively eliminate precancerous lesions. Here the inventors employ technologies to generate libraries of functionally defined stem cells of endoscopically selected, patient-matched esophageal adenocarcinoma (EAC) and its precursor lesions. Clones from these libraries meet all stem cell criteria, maintain their lesional identity, and display a remarkable and unexpected genome stability at the level of copy number and single nucleotide variation. The high-resolution phylogenetic analysis enabled by these clones defines a successively diminishing mutational threshold for transitions between indolent precursors, a discrete, “advanced” Barrett's, dysplasia, and cancer. Importantly, drug combinations that selectively eliminate Barrett's stem cells derived from multiple patients show similar efficacy against stem cells of “advanced” Barrett's, as well as dysplasia and EAC, suggesting the potential of exploiting indolent precursor lesions to identify common lineage vulnerabilities in more proliferatively aggressive lesions.
Methods
[0304] In vitro stem cell cloning from patient-matched endoscopic biopsies. Under informed consent and IRB-approved protocols at the MD Anderson Cancer Center (IRB 5 IRB00006023; LAB01-543) and the University of Connecticut Health Sciences Center (16-065-03), the inventors obtained therapy-naive samples of esophageal adenocarcinoma (EAC) and its precursor lesions. Cases 1 and 2 were from 1 mm endoscopic biopsies of adjacent lesions deemed to be EAC, Dysplasia, and Barrett's together with normal esophageal mucosa. Tissue from Case 3 was in the form of lung metastases from a primary EAC obtained in pleural effusions. Biopsies or pleural effusion cells were dissociated to single cells as described.sup.27,28 by digestion in 1 mg/ml collagenase type IV (Gibco, USA) at 37° C. for 30-45 min with agitation. Dissociated cells were passed through a 70 μm Nylon mesh (Falcon, USA) to remove aggregates, washed five times in cold F12 media, and seeded onto a feeder layer of lethally irradiated 3T3-J2 cells in StemECHO media (Multiclonal Therapeutics, Hartford, Conn., USA).sup.28 and grown at 37° C. in a 7.5% CO.sub.2 incubator with media change every 2 days. Colonies appearing in 10 days were digested by TrypLE Express solution (Gibco, USA) for 10-15 min at 37° C. and cell suspensions were passed through 30 μm filters (Miltenyi Biotec, Germany) before passaging onto new feeder lawns. Single cell cloning was performed by fine tip pipetting or by flow sorting into 384-well plates previously seeded with irradiated 3T3-J2 cells.
[0305] Stem cell differentiation. Air-liquid interface (ALI) cultures was used to assess stem cell differentiation potential.sup.27. Transwell inserts (Corning Incorporated, USA) were coated with 20% Matrigel (BD biosciences, USA) and incubated at 37° C. for 10 min to polymerize. 200,000 irradiated 3T3-J2 cells were seeded to each Transwell insert and incubated at 37° C., 7.5% CO.sub.2 incubator overnight. QuadroMACS Starting Kit (LS) (Miltenyi Biotec, Germany) was used to purify the stem cells by removal of feeder cells. 300,000 stem cells were seeded into each Transwell insert and cultured with stem cell media. At confluency (5 days), the apical media on the inserts was removed through careful pipetting and the cultures were continued in differentiation media (stem cell media without nicotinamide) for an additional 8-14 days prior to harvesting. The differentiation media was changed every one or two days.
[0306] Xenografts in immunodeficient mice. All animal experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocol 16-002 at the University of Houston. Three million stem cells were kept on ice and mixed well with 50% Matrigel (Becton Dickinson, Palo Alto, USA) to a volume of 150 μl and injected subcutaneously in NSG (NODscid IL2ra.sup.null) mice (Jackson Laboratories, Bar Harbor, USA). Xenograft size was measured with calipers and the volume was determined by the following formula: tumor volume (mm.sup.3)=½×A (mm)×B.sup.2 (mm.sup.2), where ‘A’ represent the largest dimension and ‘B’ indicates the smallest dimension.
[0307] Histology and staining. Histology, Hematoxylin and eosin (H&E) staining, Rhodamine staining, Alcian blue staining (VECTOR, USA) and immunofluorescence staining were performed using standard techniques. For immunofluorescence, 4% paraformaldehyde-fixed, paraffin embedded tissue slides were subjected to antigen retrieval in citrate buffer (pH 6.0, Sigma-Aldrich, USA) at 120° C. for 20 min, and a blocking procedure was performed with 5% bovine serum albumin (BSA, Sigma-Aldrich, USA) and 0.05% Triton X-100 (Sigma-Aldrich, USA) in DPBS(−) (Gibco, USA) at room temperature for 1 hour and then immunostained with primary antibodies at 4° C. overnight. The sources of primary antibodies used in this study include: rabbit monoclonal Ki67 (1:500, ab16667, Abcam), rabbit polyclonal Laminin (1:500, ab11575, Abcam), mouse monoclonal Cdh17 (1:300, SC74209, Santa Cruz Biotechnology), goat polyclonal E-Cadherin(1:500, AF648, R&D Systems). All images were captured by using the Inverted Eclipse Ti-Series (Nikon, Japan) microscope with Lumencor SOLA light engine and Andor Technology Clara Interline CCD camera and NIS-Elements Advanced Research v.4.13 software (Nikon, Japan) or LSM 780 confocal microscope (Carl Zeiss, Germany) with LSM software. Bright field cell culture images were obtained on an Eclipse TS100 microscope (Nikon, Japan) with Digital Sight DSFi1camera (Nikon, Japan) and NIS-Elements F3.0 software (Nikon, Japan).
[0308] DNA content analysis. Stem cells were harvested and washed twice with cold phosphate-buffered saline (PBS). After fixation in 70% cold ethanol at −20° C. for at least 1.5 h, the samples were stained using Propidium Iodide Flow Cytometry Kit (ab139418) and then analyzed by SH800 FACS Cell Sorter (Sony, Japan).
[0309] Whole exome sequencing. For exome capture and high-throughput sequencing, about 1 ug of genomic DNA was extracted using QIAGEN kits. The genomic DNA was sheared, end-repaired, A-tailed, adaptor-ligated, and Exome captured using Agilent SureSelect Human All Exon V6 Kit (Agilent Technologies, CA, USA) following the manufacturer's recommended protocols. In short, fragmentation was conducted by hydrodynamic shearing system (Covaris, Massachusetts, USA) to generate 180-280 bp fragments. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, adapters were ligated. Fragments with ligated adapters on both ends were selectively enriched in a PCR reaction. Captured libraries were enriched in a PCR reaction to add indexes to prepare for hybridization. Products were purified using AMPure XP system (Beckman Coulter, Beverly, USA) and quantified with the Agilent high-sensitivity DNA assay on the Agilent Bioanalyzer 2100 System. The multiplexed libraries were sequenced on Illumina HiSeq X platform (150 bp paired-end reads, Illumina, California, USA). The clusters that do not pass the Chastity filter were removed from downstream analysis. At least 20 million paired reads were generated for each sample.
[0310] Low-pass whole genome sequencing. Sequencing libraries were prepared by TruSeq Nano DNA HT Sample Prep Kit (Illumina, California, USA) following the manufacturer's protocol. First, 1000 ng of genomic DNA was fragmented by sonication to 350 bp. Then fragments were end-repaired, A-tailed and adaptor-ligated, followed by further PCR reactions. After purification using the AMPure XP system (Beckman Coulter, Beverly, USA), the library was size-selected using Agilent 2100 Bioanalyzer and quantified by real-time PCR. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using Hiseq PE Cluster Kit (Illumina, California, USA) according to the manufacturer's standard protocol. Next, the libraries were sequenced on Illumina Hiseq X platform (Illumina, California, USA) in 150 bp paired-end model. At least 20 million paired-end reads were generated for each sample.
[0311] SNV/Indel/CNV and ploidy calling. Data preprocessing. The raw sequencing reads were quality controlled by removing the adapters' bases and the low-quality bases (Phred-value<10) from the read ends and by discarding the reads with >10% ambiguous bases inside using Trimmomatic.sup.51 version 0.36. Murine sequences were filtered using xenome.sup.52 version 1.0.1 with default parameters. The remaining reads were aligned to human reference genome (UCSC hg19) using BWA.sup.53 version 0.7.15-r11403 under the mem model requiring map quality >=40 (Phred-value). PCR duplicates were removed using Picard tool version 2.15.0 (broadinstitute.github.io/picard/). The GATK.sup.54,55 version 3.8.04 was used to realign the reads near indels (Mills_and_1000G_gold_standard indels bundled within GATK pipeline) and to recalibrate the base qualities with default settings following the best practice protocol.sup.55.
[0312] SNVs/Indels calling. SNVs and Indels were called by software Manta.sup.56 version 1.3.25 and Strelka.sup.57,58 version 2.9.26 with default parameter values in somatic calling model. Only the SNVs/Indels that passed the default filter of Manta and Strelka in derived vcf files were used in downstream analyses. The inventors also applied harder filters to the variants that require only two genotypes presence, variant quality (Phred-value)>30, total read depth >15, alternative allele depth >5, and alternative allele proportion >5%. Also, they required that the corresponding matched normal sample were homozygous wild type at the mutation sites. Somatic mutations were further filtered to remove possible germline mutations based on a panel of 27 normal samples. Somatic mutations with allele frequencies less than 0.01 in 1000 Genome database or gnomAD database were discarded as well. SNVs and Indels were annotated with ANNOVAR web version.
[0313] CNV calling. The GATK.sup.59 somatic copy number variants calling pipeline version 4.0.4.0 (gatkforums.broadinstitute.org/gatk/discussion/9143/) was used to call the CNVs. The inventors used 17 normal female samples sequenced on the same platform to build the CNV panel of normals (PoN) with extra parameter “—minimum-interval-median-percentile 10.0”. The contigs shorter then 46709983 bp were excluded for further analysis. The 1000G phase1 high-quality SNPs (1000G_phase1.snps.high_confidence bundled within GATK pipeline) was used to collect allelic counts information. In the segmentation step, the inventors used patient-matched normal samples and applied parameters “—number-of-smoothing-iterations-per-fit 1—minimum-total-allele-count 15—window-size 7500”. The other steps used the default settings. Segments with less than 15 SNVs were excluded. After getting the segmented confidence interval of copy ratio and allele fraction information, the absolute allelic copy number was inferred and curated manually by considering the consistence of copy ratio and allele fractions and the unique features in different copy number (CN) patterns (e.g., CN1's allele fraction=0 or 1, CN3's allele fraction=0.33 or 0.66). Briefly, after generating segmented copy-ratio (sCR) and allele-fraction (sAF) results from GATK pipeline, the inventors first determined the absolute copy number for CN0 (copy number=0), CN1, and CN2 regions by genome-wide analysis of the raw sequencing reads at germline heterozygous sites in the respective Barrett's, Dysplasia, and EAC clones based on the expectation that CN0 regions have no sequencing reads regardless of amplification of surrounding regions, CN1 regions are homozygous, and some of the CN2 regions show heterozygosity. Then, based on the evenly distributed copy-ratio peaks and clearly separated confidence intervals of each peak, as well as its consistency with sAF patterns, the inventors assigned absolute copy number integers to CN3, CN4, CN5, etc. For instance, with Dysplasia clone D1-5, considering even spacing of sCR patterns (close to 0.35), they assigned CN0 to position R5 lacking reads, CN1 to R8 due to homozygosity (sCR=0.35, sAF=0.00/0.99), CN2 to R6 on account of heterozygosity (sCR=0.71, sAF=0.48/0.51), CN3 to R2 (sCR=1.05, sAF=0.32/0.67), CN4 to R1 (sCR=1.34, sAF=0.24/0.75), CN5 to R9 (sCR=1.71, aAF=0.40/0.59. The CNV result was further confirmed by ABSOLUTE.sup.37 algorithm with default parameters. In selecting the best model of ABSOLUTE output, the inventors required that each copy number peak should be under an integral number, the bottom peak for copy number should be close to zero, and the ploidy value should be very close (>0.95) to 1 because the data were obtained from single cell-derived clones. The top model meeting these criteria was considered as the best model.
[0314] Ploidy calling. After getting the copy number profiles, the inventors multiplied each segment's absolute copy number to its proportion of the genome in length and added up the derived products as the ploidy number.
[0315] Phylogenetic tree construction and ordering of somatic mutations. Ternary genotypes of filter-passed somatic SNVs identified from all WES data by Strelka were used in phylogenetic tree construction. The genotypes of normal sample (e.g., matched blood or fibroblast) were added as an outgroup. The trees were built by SiFit.sup.60 that employs a heuristic search algorithm to infer the Maximum Likelihood (ML) phylogenetic tree under a finite-site model of evolution. The number of iterations was set to 10000. The “InferAncestralStates” program of SiFit was used for inferring the order of somatic mutations on the branches of the phylogeny based on the false negative rate, deletion rate, and LOH rate reported by SiFit during learning the tree in tree building step.
[0316] Clonality analysis. To ensure that each pedigree was single cell-derived, the inventors analyzed the distribution of variant allele fractions (VAFs) of the identified somatic mutations in each sample. For monoclonal pedigrees, diploidy (2n) pedigrees' VAFs should distribute around 50% and triploidy (3n) pedigree will have VAFs around 0.33 and 0.66. For polyclonal pedigrees, most of somatic mutations should have VAFs less than 0.5. Polyclonal pedigrees were excluded from further analysis.
[0317] Expression analysis. Total RNAs were extracted from immature stem cell colonies for microarray analysis. RNAs were amplified using WT Pico RNA Amplification System V2 and Encore Biotin Module (NuGEN Technologies, CA, USA). All samples were prepared according to manufacturer's instructions and hybridized onto GeneChip Human Exon 1.0 ST array (Affymetrix, CA, USA). GeneChip operating software was used to process all the Cel files and Affymetrix Expression Console software was used for quality control analysis of microarray data. The gene expression analysis was performed using Partek Genomics Suite 6.6 (Partek Incorporated, USA). All the probe intensity values were normalized and log 2-transformed. To identify the differentially expressed genes, 1-way ANOVA was performed (cutoff value: log.sub.2 fold-change >1.5 and p<0.05). All the comparisons were performed as a pairwise manner and gene sets from each comparison were overlapped and selected the unique gene signature for each sample. Unsupervised clustering and heatmap generation were performed with sorted datasets by Euclidean distance based on average linkage clustering, and Principal Component Analysis (PCA) map was made using all probe sets. Pathway enrichment analysis was performed using Enrichr.sup.61.
Results
[0318] Clonogenic cells from patient-matched lesions. A series of 1 mm endoscopic biopsies from adjacent regions of Barrett's, dysplasia, and esophageal adenocarcinoma was obtained from therapy-naive patients suspected of early esophageal adenocarcinoma (
[0319] Interclonal heterogeneity and clonal genomic stability. To assess clonal heterogeneity within and across these lesion-specific stem cell libraries, the inventors selected 76 single cell-derived clones from Case 1 (6 esophageal, 20 Barrett's, 19 dysplasia, and 32 EAC) for expansion and low-pass, whole-genome sequencing (lpWGS; ave. 1.6× coverage;
[0320] While these clones seemed to accurately reflect the mutational profiles of the neoplastic cells in the patient biopsies, it was less clear whether the known genomic instability of cancers.sup.32-34 would, over extended growth, degrade the proxy value of these clones. In this regard, the inventors noted that some of the dysplasia clones and all EAC clones displayed a chromothripsis event of chromosome 16 marked by complex rearrangements and translocations (e.g.,
[0321] Clonal phylogenetics to cancer. To assess the evolutionary relationships between the Barrett's, Dysplasia, and EAC clones, the inventors performed a phylogenetic analysis across the 35 clones with WES data of Case 1 based on 679 somatic SNVs (allele frequency >0.2) from the WES data of the 35 clones (
[0322] From a second case of esophageal adenocarcinoma, the inventors determined the phylogenetic relationships between 45 clones sampled from libraries based on 463 somatic SNVs (
[0323] BE2 and thresholds in oncogenesis. While detailed clonogenic analyses were limited to two cases of EAC, the parallels seen in the transitions from BE1, BE2, DYS, and EAC clones suggest patterns that likely have clinical correlates (
[0324] Aside from low-risk Barrett's esophagus and high-risk dysplasia, there is much clinical interest in the presence of histological intermediates known as “low-grade dysplasia (LGD)” and “indeterminant for dysplasia” as a harbinger for progression. While the inter-observer agreement for LGD can be low, there is general agreement that LGD has an enhanced risk (0.4-13.4%/year) for progression to dysplasia and EAC.sup.43,44. The BE2 clones identified from both EAC cases examined here display a partial loss of polarity upon differentiation in 3-D cultures consistent with LGD, and show a mutational profile (p53 mutations, ERRB2 amplifications or mutational activation, multiple CAMs and CNV events) that would conceivably enhance its risk for further progression over BE1. To identify BE2 biomarkers that could aid in the detection of LGD, the inventors compared whole genome expression profiles of 3-D epithelia formed by BE1 (BE1-5) and BE2 (BE2-8) clones. A volcano plot of these data indicates that BE1 epithelia express known markers of Barrett's esophagus (e.g., TFF1, TFF2, and TFF3, SPINK1 and SPINK4, and CLDN18), whereas BE2 epithelia express an array of genes, exclusive of those in amplified loci, including NRCAM, CEACAM6, CDH17, PTPRS, and FABP1, among many others. The inventors anticipate that a panel of such biomarkers could aid in the detection of BE2 clones in a field of BE1 clones to stratify risk in patients with Barrett's esophagus.
[0325] Small molecule screens against precursor stem cells. Given that Barrett's esophagus is an essential precursor for EAC, the inventors adapted BE1 stem cells to high-throughput screening platforms to identify proof-of-concept leads for preemptive therapies. Parallel screens of BE1 and normal esophageal stem cells in 384-well plates against small molecule collections yielded off-diagonal a set of off-diagonal nominal hits of which many showed differential lethality against BE1 stem cells. However, the best of these molecules showed upon dose-response assays to have an 20-fold IC.sub.50 advantage over normal esophageal stem cells. In the process of screening, the inventors noted several compounds that enhanced the growth of the normal esophageal stem cells while marginally inhibiting the growth of the target BE1 stem cells. Across screens of Barrett's esophagus stem cells from eight cases, the inventors identified the tyrosine kinase inhibitor ponatinib.sup.45 as the best of these esophageal stem cell “promoters”. The inventors rescreened the BE1 and normal esophageal stem cells in the presence of ponatinib to yield a new set of hits that effectively inhibited BE1 stem cells while sparing the esophageal stem cells. One of these, SM-164, is an inhibitor of XIAP, one of a set of 8 IAP proteins known to regulate caspase-mediated cell death.sup.47. In combination with ponatinib, SM-164 effectively eliminates BE1 stem cells with an IC.sub.50 of less than 1 nM with minimal impact on normal esophageal stem cells. In co-cultures of BE1 and normal esophageal stem cells that potentially mimic the interactions between these cells in the distal esophagus, this drug combination selectively eliminates the BE1 cells while promotes the expansion of the normal esophageal stem cells.
[0326] Given the efficacy of the SM-164/ponatinib combination against BE1 stem cells, the inventors asked if it would have any effect on stem cells of more advanced BE2, DYS, and EAC lesions. Remarkably, the combination showed similar efficacy against the entire lineage of BE1 to EAC even though these compounds were identified for their effect on BE1.
Discussion
[0327] The present work applied technology for single cell cloning of normal mucosal stem cells to multiple, patient-matched lesions implicated in the oncogenesis of esophageal adenocarcinoma. The salient features of the cells cloned from these lesions, including high clonogenicity, unlimited proliferative capacity, and absolute fate commitment to the respective BE1, BE2, DYS, and EAC lesions both in vitro and in vivo, generalizes the cancer stem cell concept to all lesions in oncogenesis.sup.15,46,47. The clonal analysis afforded by these cells demonstrates that the vast majority of single nucleotide and copy number variation events present in these clones preexisted in the patient's lesion and were not a consequence of the adaptation of these cells to culture. Moreover, tracking individual DYS and EAC clones through extensive propagation accompanying serial passaging in vitro and clonal tumors in vivo reveal their immense and unexpected genomic stability at both CNV and SNV levels. These features enabled a high-resolution assembly of the phylogenetic relationships between these patient-matched precursor lesions and the presenting tumor which likely evolved over years and even decades. In the two EAC cases assessed in detail, this analysis revealed a discrete clade of stem cells that evolved from BE1 and gave rise to DYS clones. This intermediate, termed “BE2” was distinguished from the BE1 clones by the loss of p53 and the gain of ERBB2 activity, in addition to a host of other single nucleotide and copy number variation events, and likely corresponds to the clinical entity of “low-grade dysplasia” associated with enhanced risk for progression to high-grade dysplasia and EAC.sup.1,43,44. The inventors' comparison of the gene expression profiles of these BE1 and BE2 clones has identified a common panel of genes across these two patients whose expression could assist in the identification of patients with Barrett's esophagus who are at risk for progression to dysplasia and EAC. An examination of the in-line mutational profiles across the BE1, BE2, DYS, and EAC clades revealed major changes from BE1 to BE2 and from BE2 to DYS, but very minimal changes from DYS to EAC, the latter amounting to a small number of new code-altering mutations and few or no CNV events. Overall, the magnitude and specificity of the mutational profiles in each of the transitions argues that once the BE2 stage is achieved, the subsequent transitions to DYS and to EAC seem progressively more likely. These findings support the early screening for Barrett's and especially BE2, as well as the development of therapeutics that target the discrete stem cell populations of these lesions. Lastly, the stem cells cloned in this work are likely essential for the future regenerative growth of the lesions from which they were derived, and therefore represent fitting targets for both preemptive and post facto therapeutics.
[0328] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
REFERENCES
[0329] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
References for Results and Discussion
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